Improving umbilical cord blood processing to increase total nucleated cell count yield and reduce cord input wastage by managing the consequences of input variation.

BACKGROUND AIMS: With the rising use of umbilical cord blood (UCB) as an alternative source of hematopoietic stem cells, storage inventories of UCB have grown, giving rise to genetically diverse inventories globally. In the absence of reliable markers such as CD34 or counts of colony-forming units, total nucleated cell (TNC) counts are often used as an indicator of potency, and transplant centers worldwide often select units with the largest counts of TNC. As a result, cord blood banks are driven to increase the quality of stored inventories by increasing the TNC count of products stored. However, these banks face challenges in recovering consistent levels of TNC with the use of the standard protocols of automated umbilical cord processing systems, particularly in the presence of input variation both of cord blood volume and TNC count, in which it is currently not possible to process larger but useable UCB units with consequent losses in TNC. METHODS: This report addresses the challenge of recovering consistently high TNC yields in volume reduction by proposing and validating an alternative protocol capable of processing a larger range of units more reliably. RESULTS: This work demonstrates improvements in plastic ware and tubing sets and in the recovery process protocol with consequent productivity gains in TNC yield and a reduction in standard deviation. CONCLUSIONS: This work could pave the way for cord blood banks to improve UCB processing and increase efficiency through higher yields and lower costs.

Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts.

BACKGROUND: Conditions for maintaining hematopoietic progenitor cells (HPCs) before cryopreservation remain controversial. An understanding of the impact of time and temperature during nonfrozen storage can contribute to the maintenance of the quality of products, improving transplantation outcomes. The objective of this study was to determine the influence on cell potency of thawed products from three sources of HPCs after prolonged storage at different temperatures before cryopreservation. STUDY DESIGN AND METHODS: Viable cell counts by flow cytometry and colony-forming unit (CFU) recoveries were assessed on cord blood (CB), mobilized peripheral blood stem cell (PBSC), and bone marrow (BM) samples over 72 hours using two different storage conditions, refrigerated (4-8°C) or room temperature (19-22°C). To determine the effects of delayed freezing on progenitor recoveries, paired samples were evaluated before and after cryopreservation. RESULTS: All samples maintained at refrigerated temperatures resulted in higher recoveries than those at room temperature in all variables assessed. Specifically, when assessing for CFU yields after thawing, the impact of time on BM resulted in a significant loss as soon as 24 hours (n = 10, 36.4 ± 28.0%, p = 0.003). This decrease was also observed for PBSCs and CB but at 48 hours of fresh storage (PBSCs n = 11, 32.7 ± 26.2%, p = 0.006; CB n = 10, 39.6 ± 26.4%, p = 0.001). CONCLUSION: Our data suggest that HPC products are better maintained at refrigerated temperatures before cryopreservation. Delaying cryopreservation should be minimized to avoid significant losses in cell potency.

Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment.

BACKGROUND: Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions. STUDY DESIGN AND METHODS: To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach. RESULTS: Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration. CONCLUSION: Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.

Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton's Jelly.

BACKGROUND: The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards.

Adapting Cord Blood Collection and Banking Standard Operating Procedures for HLA-Homozygous Induced Pluripotent Stem Cells Production and Banking for Clinical Application.

In this article, we will discuss the main aspects to be considered to define standard operation procedures (SOPs) for the creation of an induced pluripotent stem cell (iPSC) bank using cord blood (CB)-or similar cell type-bank guidelines for clinical aims. To do this, we adapt the pre-existing SOP for CB banking that can be complementary for iPSCs. Some aspects of iPSC manufacturing and the particular nature of these cells call for special attention, such as the potential multiple applications of the cells, proper explanation to the donor for consent of use, the genomic stability and the risk of genetic privacy disclosure. Some aspects of the iPSC SOP are solidly established by CB banking procedures, other procedures have good consensus in the scientific and medical community, while others still need to be further debated and settled. Given the international sharing vocation of iPSC banking, there is an urgent need by scientists, clinicians and regulators internationally to harmonize standards and allow future sample interchange between many iPSC bank initiatives that are springing up worldwide.

In vitro toxicity of three new antitumoral drugs (trabectedin, aplidin, and kahalalide F) on hematopoietic progenitors and stem cells.

OBJECTIVE: In addition to neutropenias and/or thrombocytopenias as a short-term effect, antineoplastics also can produce long-term effects as a consequence of damage to the hematopoietic stem cells. The aim of the present study was to evaluate the toxicity of three marine-derived antineoplastics on murine hematopoietic stem cells. These antitumoral compounds currently are being evaluated in patients in phase II (aplidin and kahalalide F) and phase II/III (trabectedin) clinical trials. MATERIALS AND METHODS: Long-term competitive repopulating assays were performed in mice to analyze toxic effects on the hematopoietic stem cells responsible for the multipotential long-term repopulation of hematopoiesis. Furthermore, granulocytic and T- and B-lymphoid lineages were studied, as well as myeloid (CFU-GM) and megakaryocytic (CFU-Meg) progenitors. RESULTS: When cells were treated in vitro for 24 hours with CFU-GM IC(50) dose of trabectedin (9.59+/-4.96 nM), no significant effects were observed in the stem cells. The dose of trabectedin that produced 90% of inhibition in CFU-GM (IC(90): 23.71+/-1.27 nM) only inhibited 45% survival of stem cells. Doses of aplidin that produced reductions of 50% (56.9+/-13.32 nM) or 90% (195.88+/-21.39 nM) in myeloid progenitors did not show any effect on hematopoietic stem cells. Kahalalide F did not show any toxic effect in either short-term or long-term repopulating cells up to 10 microM. CONCLUSIONS: Our data show that the hematopoietic stem cells effects of antitumoral drugs can be properly characterized by the murine competitive repopulating assays. Our results suggest that long-term myelosuppression as a consequence of trabectedin, aplidin, or kahalalide F treatment would not be expected.

Regulatory elements of the vav gene drive transgene expression in hematopoietic stem cells from adult mice.

OBJECTIVE: Previous studies have shown that the HS21/45 promoter of the vav protooncogene drives a predominant expression of exogenous transgenes in mouse hematopoietic cells, including clonogenic bone marrow (BM) progenitors. We investigated the activity of this promoter in the hematopoietic stem cell compartment of adult mice. MATERIALS AND METHODS: Inbred Ly5.1 transgenic mice expressing a nonfunctional human CD4 marker gene (hCD4) under the control of the HS21/45 promoter were generated. BM cells from these animals were sorted based on the intensity of hCD4 expression. Fractions characterized by high, intermediate, or low/negative expression of the transgene were then assessed for their competitive repopulation ability (CRA), using unfractionated BM cells from Ly5.2 mice as a reference competitor population. RESULTS: Data showed that BM cells having a low/negative or intermediate expression of hCD4 had a very poor hematopoietic CRA. In contrast, BM cells with high hCD4 expression were characterized by a high CRA. These observations were confirmed in the short- and long-term posttransplantation of primary and secondary recipients when analyzing the lymphoid and myeloid cells of recipient mice. CONCLUSIONS: Our results demonstrate for the first time that the regulatory HS21/45 sequence of the vav gene constitutes an efficient promoter for driving transgene expression in multipotent hematopoietic stem cells residing in the BM of adult mice. Thus, this promoter is proposed for the development of transgenic mice and gene therapy vectors that require restricted expression of exogenous transgenes in cells of the hematopoietic system, including primitive hematopoietic stem cells.

Use of CFU-GM assay for prediction of human maximum tolerated dose of a new antitumoral drug: Yondelis (ET-743).

Acute cytotoxic exposure causes decreases in bone marrow progenitors that precedes the neutrophil nadir. Experiments in animal models reveal a relationship between the reduction in granulocyte-macrophage progenitors (CFU-GM) and the decrease in absolute neutrophil count [Toxicol. Pathol. 21 (1993) 241]. Recently, the prevalidation of a model for predicting acute neutropenia by the CFU-GM assay has been reported [Toxicol. In Vitro 15 (2001) 729]. The model was based on prediction of human MTD by adjusting the animal-derived MTD for the differential sensitivity between CFU-GM from animal species and humans. In this study, this model has been applied on a new antitumoral drug, Yondelis (Ecteinascidin; ET-743). Preclinical studies showed that hematotoxicity was the main side effect in mice, being the MTD of 600 microg/m2 [Drugs Future 21 (1996) 1155]. The sensitivity of myeloid progenitors was higher in mice than in humans, with IC90 values of 0.69+/-0.22 nM and 1.31+/-0.21 nM for murine and human CFU-GMs respectively. This study predicts a human MTD of 1145 microg/m2. The reported human MTD of ET-743 given as a 24-h continuous infusion every 3 weeks is 1800 microg/m2 [J. Clin. Oncol. 19 (2001) 1256]. Since our predicted MTD is within fourfold of the actual MTD (the interspecies variation in tolerated dose due to differences in clearance rates, metabolism pathways and infusion rate) the result confirms the profit of the prediction model.

Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.

A simpler method of preprocessing MALDI-TOF MS data for differential biomarker analysis: stem cell and melanoma cancer studies.

INTRODUCTION: Raw spectral data from matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) with MS profiling techniques usually contains complex information not readily providing biological insight into disease. The association of identified features within raw data to a known peptide is extremely difficult. Data preprocessing to remove uncertainty characteristics in the data is normally required before performing any further analysis. This study proposes an alternative yet simple solution to preprocess raw MALDI-TOF-MS data for identification of candidate marker ions. Two in-house MALDI-TOF-MS data sets from two different sample sources (melanoma serum and cord blood plasma) are used in our study. METHOD: Raw MS spectral profiles were preprocessed using the proposed approach to identify peak regions in the spectra. The preprocessed data was then analysed using bespoke machine learning algorithms for data reduction and ion selection. Using the selected ions, an ANN-based predictive model was constructed to examine the predictive power of these ions for classification. RESULTS: Our model identified 10 candidate marker ions for both data sets. These ion panels achieved over 90% classification accuracy on blind validation data. Receiver operating characteristics analysis was performed and the area under the curve for melanoma and cord blood classifiers was 0.991 and 0.986, respectively. CONCLUSION: The results suggest that our data preprocessing technique removes unwanted characteristics of the raw data, while preserving the predictive components of the data. Ion identification analysis can be carried out using MALDI-TOF-MS data with the proposed data preprocessing technique coupled with bespoke algorithms for data reduction and ion selection.